Website address: www.niscair.res.in

 

Indian Journal of Biochemistry & Biophysics

Total visitors: 4,171  since 11-04-07

CODEN : IJBBBQ  ISSN : 0301-1208

 

VOLUME 44

NUMBER 2

APRIL 2007

 
CONTENTS

 

 

Papers

 

Expression, purification and characterization of a synthetic gene encoding human amyloid ß (Aß1-42) in Escherichia coli

71

        Sarada Subramanian* and A N Divya Shree

 

 

 

Purification and properties of α-galactosidase from white-rot fungus Pleurotus florida

76

        Ramalingam*, N Saraswathy, S Sadasivam, K Subha and N Poorani

 

 

 

Electrochemical studies of glucose oxidase immobilized on glutathione coated gold nanoparticles

82

        Sridevi Akella and Chanchal K Mitra*

 

 

 

Improved method for estimation of inorganic phosphate: Implications for its application in enzyme assays

88

        Samir P Patel, Minal A Patel, Hiren R Modi* and Surendra S Katyare

 

 

 

Role of pepsin in modifying the allergenicity of Bhetki (Lates calcarifer) and Mackerel (Rastrelliger kanagurta) fish

94

        Gautam Mondal, Urmimala Chatterjee, Soumya Samanta and
Bishnu P Chatterjee*

 

 

 

Studies on interactions between plant secondary metabolites and glutathione transferase using fluorescence quenching method

101

        Xian Zhang, Xinsheng Cheng*, Chuanqin Wang, Zechun Xue, Liwen Yang and Zheng Xi

 

 

 

Construction and design of single stranded collagen-like structure

106

        Fateh Singh Nandel* and Avneet Saini

 

 

 

Exploring QSAR of peripheral benzodiazepine receptor binding affinity of N,N-dialkyl-2-phenylindol-3-yl-glyoxylamides using physico-chemical descriptors

114

        Kunal Roy* and Manoj Kumar Dalai

 

 

 

Note

 

Purification of protein from a crude mixture through SDS-PAGE transfer method

122

        Dipankar Bhattacharyya, Arindam Basu and Parimal C Sen*

 

 

 

Instructions to Authors

126

 

——————

*Author for correspondence

Papers

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, April 2007, pp 71-75

 

 

Expression, purification and characterization of a synthetic gene encoding human amyloid ß (Aß1-42) in Escherichia coli

Sarada Subramanian* and A N Divya Shree

Department of Neurochemistry, National Institute of Mental Health & Neurosciences, Bangalore 560 029, India

Received 08 December 2006 ; revised 28 February 2007

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive function. Existing evidence indicates that abnormal processing and extracellular deposition of the longer form of the amyloid peptide Aß(1-42), a proteolytic derivative of the amyloid precursor protein (APP), is a key step in the pathogenesis of AD. Active immunization with Aß(1-42) has been shown to decrease brain Aß deposition and improve cognitive performance in mouse models of AD. In the present study, we sought to express the synthetic gene encoding Aß in Escherichia coli to enable rapid production of the antigen and its purification. The synthetic gene has been constructed from six oligonucleotides by employing overlapping PCR strategy and expressed in E. coli using the T7 promoter system. The recombinant peptide has been purified to homogeneity by a single step Ni+2 affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) using polyclonal anti-Aß(1-42) sera confirms that the corresponding linear B-cell epitopic sequences are available for immunorecognition in the recombinant peptide. This methodology enables rapid, continuous production and purification in bulk amounts of human Aß sequence by employing bacterial expression system

Keywords: Alzheimer’s disease, Amyloid ß peptide, Escherichia coli, Overlapping PCR, Synthetic gene, ELISA, Expression, Purification and characterization

*E-mail: sarada@nimhans.kar.nic.in; saradabs@yahoo.com

 

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, April 2007, pp. 76-81

 

Purification and properties of α-galactosidase from
white-rot fungus Pleurotus florida

Ramalingam*, N Saraswathy, S Sadasivam, K Subha and N Poorani

Department of Biotechnology, Kumaraguru College of Technology, Coimbatore 641 006, Tamil Nadu, India

Received 26 June 2006; revised 15 February 2007

a-Galactosidase was strongly induced in the white-rot fungus Pleurotus florida by arabinose than its natural substrates and was purified to homogeneity by acetone precipitation, ultrafiltration and DEAE-Sepharose chromatography. The enzyme was a monomeric protein with a molecular mass of @ 99 kDa, as revealed by native-PAGE and SDS-PAGE. a-Galactosidase was optimally active at 55°C for the hydrolysis of p-nitrophenyl-a-galactopyranoside (PNPaG) and lost its 20% and 50% of original activity in 30 min at 60°C and 70°C, respectively. The pH optimum of the enzyme was between 4.6 and 5.0. It was stable in a wide pH range (pH 4.0 to 9.0) at 55°C for 2 h. The Ag+ and Hg2+ strongly inhibited the enzyme activity. Galactose, glucose, maltose and lactose also inhibited the enzyme activity, whereas N-bromosuccinimide treatment resulted in near total loss of acitivity. The Km and Vmax values of the enzyme for PNPaG were found to be 1.1 mM, and 77 mmol min-1 mg-1, respectively. a-Galactosidase immobilized in agar was more effective for the degradation of raffinose than in the sodium alginate. TLC results indicated its potential for the removal of raffinose and stachyose in soymilk.

Keywords: Pleurotus florida, a-Galactosidase, Purification, Kinetic studies, Soymilk, Immobilization, Agar

*E-mail: gobiram@rediffmail.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, April 2007, pp. 82-87

 

Electrochemical studies of glucose oxidase immobilized on glutathione coated gold nanoparticles

 

Sridevi Akella1 and Chanchal K Mitra2*

School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India

Received 06 November 2006 ; revised 15 March 2007

Glutathione (L-g -glutamyl-L-cysteinyl-L-glycine; GSH) forms a surface monolayer on gold nanoparticles by tethering via sulfur bonds (Au:GSH). In the present study, glucose oxidase (GOx; EC 1.1.3.4) was immobilized by covalent chemical coupling reactions on to Au:GSH nanoparticles and the enzyme coupled nanoparticles formed a stable colloid (stable for several weeks) in water. The immobilized enzyme was investigated for electrochemical characteristics to monitor the FAD (prosthetic group of the GOx) redox potentials. Various concentrations of substrate (glucose) were added to check the oxidation characteristics. It was observed that with increase in substrate concentrations, the oxidation rate increased proportionally with the current. The present study demonstrated that GOx was effectively coupled to the gold nanoparticle (Au:GSH). The coupled nanoparticle system could be used in a potential biosensor application. Similarly, other enzymes (e.g., horseradish peroxidase) could be immobilized to the Au:GSH nanoparticles via the peptide arm (GSH) to achieve the desired characteristics needed for a specific application in biosensor.

Keywords: Glutathione, Glucose oxidase, Gold nanoparticle, Biosensor

 

         E-mail: 1neuron555@yahoo.co.in; 2c_mitra@yahoo.com

 

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, April 2007, pp. 88-93

 

Improved method for estimation of inorganic phosphate: Implications
for its application in enzyme assays

Samir P Patel§, Minal A Patel, Hiren R Modi* and Surendra S Katyare

Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat 390 002, India

Received 21 December 2006; revised 13 March 2007

The conventional method of Fiske and Subba Row for the estimation of inorganic phosphate (Pi) is although rapid, but suffers from the disadvantage that the color is unstable and hence the optical density (OD) measurements have to be carried out within a short time span of 8-12 min. This poses a restriction on the number of samples, which can be handled in a batch. Although, modified procedures involving use of alternate reducing agents/or increasing the concentration of H2SO4 in conventional method have been subsequently developed, but the problem of color stability could not be solved. In addition, the use of higher concentrations H2SO4 has rendered the methods unsuitable in enzyme assays, especially if the acid labile phosphate containing substrates have been used. In the present study, attempts have been made to suitably modify the method to improve the stability of the color and sensitivity and also for its applicability in enzyme assays, especially when acid labile phosphate containing substrates such as ATP is used. We used the higher concentrations (0.625, 0.8 and 1.0 N) of H2SO4 rather than 0.5 N used in the conventional assay procedures. Under these conditions, the reagent blanks do not develop color for up to 24 h, whereas the intensity of the molybdenum blue color in the standard and/or experimental tubes increased with time reaching optimum value at 24 h. Simultaneously, the absorption maximum shifts from 660 nm to 820 nm. The highest concentration of H2SO4 (1.0 N) is found to be the most effective in the process of color development. The sensitivity of the method is from 1.7 to 2.1 times higher, as compared to the conventional Fiske and Subba Row method for the measurements carried out at the end of 15 min at 820 nm and with the highest concentration of H2SO4 (1.0 N); the sensitivity increased 4.8-fold at the end of 24 h. Presence of glucose and sucrose (1-10 mM), NaCl and KCl (5-100 mM), MgCl2 (1-10 mM) and BSA (10 to 500 µg per assay tube) do not interfere either with color development or with OD measurements. The extent of ATP hydrolysis is 1.6 to 3.4% for up to 1 h, depending upon the concentration of H2SO4 used. Only negligible hydrolysis of G6P is observed under these conditions. These results suggest that the presently modified method is suitable for Pi analysis in the enzyme assays, in the presence of labile phosphate containing substrates.

Keywords: Pi determination, Stabilization, Molybdenum blue color, Improved sensitivity

 

*E-mail: modi_hiren@yahoo.co.in

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, April 2007, pp 94-100

 

Role of pepsin in modifying the allergenicity of Bhetki (Lates calcarifer) and Mackerel (Rastrelliger kanagurta) fish

Gautam Mondal, Urmimala Chatterjee, Soumya Samanta and Bishnu P Chatterjee*

Department of Biological Chemistry, Indian Association for the Cultivation of Science,
Jadavpur, Kolkata 700 032, India

Received 06 August 2006; revised 2 March 2007

The effect of pepsin digestion on the allergenicity of raw and thermally processed (boiled and fried) fish muscle extracts of two widely consumed fishes bhetki (Lates calcarifer) and mackerel (Rastrelliger kanagurta) was studied. Sere were collected from 110 patients who were hypersensitive to fish, as evidenced by their clinical history, symptoms and positive skin-prick test results. The various extracts after digestion with pepsin at different times of incubation were tested for specific IgE-binding activity by ELISA and immunoblotting with patients’ sera. All the extracts of both the fishes retained their allergenicity as evidenced by ELISA and immunoblotting. In bhetki, maximum allergenicity was found in the pepsin-digested fried extract, whereas similar treatment decreased the allergenicity in fried mackerel. Results showed that raw as well as thermally processed allergens of both the fishes maintained strong allergenicity, even after digestion with pepsin for different time periods. The study revealed that the fish proteins played an important role in manifestation of allergy, due to their stable structure, which was retained even after pepsin and heat treatment.

Keywords: Allergen, Bhetki, Mackerel, Pepsin, IgE-reactivity, ELISA, Immunoblotting

*E mail: cbishnup@gmail.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, April 2007, pp. 101-105

 

Studies on interactions between plant secondary metabolites and glutathione transferase using fluorescence quenching method

Xian Zhang, Xinsheng Cheng*, Chuanqin Wang, Zechun Xue, Liwen Yang and Zheng Xi

Department of Chemistry, University of Science & Technology of China, Hefei 230026, China

Received 22 February 2006; revised 5 March 2007

The interactions between plant secondary metabolites (tannic acid, rutin, cinnamic acid and catechin) and glutathione transferase (GST) were investigated by fluorescence and UV-Vis absorption spectroscopy. Intrinsic fluorescence of GST was measured by selectively exciting their tryptophan (Trp) residues and quenching constants were determined using the Stern-Volmer equation. The binding affinity was found to be strongest for tannic acid and ranked in the order tannic acid>rutin>cinnamic acid>catechin. The pH values in the range of 6.7-7.9, except for tannic acid, did not affect significantly the affinity of rutin, cinnamic acid and catechin with GST. Results showed that the fluorescence quenching of GST was a static quenching. Fluorescence quenching and UV-Vis absorption spectroscopy suggested that only the tannic acid changed the microenvironment of the Trp residues. Furthermore, the number of binding sites and binding constants at different pH values showed that tannic acid had strongest affinity towards GST and hydrogen bonding played an important role in the affinity between GST and the metabolites.

Keywords: Plant secondary metabolites, Glutathione transferase, Fluorescence quenching, UV-Vis absorption spectroscopy

 

*E-mail:xscheng@ustc.edu.cn

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, April 2007, pp. 106-113

 

Construction and design of single stranded collagen-like structure

Fateh Singh Nandel* and Avneet Saini

Department of Biophysics, Panjab University, Chandigarh160014, India

Received 06 October 2006; revised 10 February 2007

Polytheonamide B, a 48 residue long highly cytotoxic polypeptide extracted from marine sponges contains amino acids of alternate chirality and the N-terminal region is rich in t-Leu residues. The aim of this study is to analyze the effect of these alternate chiralities and conformational behavior of various model peptides containing t-Leu, in order to explore their role in designing bioactive peptides that shall offer advantages comparable to polytheonamide B, while circumventing its limitations. The conformational behavior of various peptides constructed from t-Leu of the form Ac-(l/d-X-l/d-Y)n-NHMe, where X = Gly/Ala/Leu and Y = t-Leu has been studied and compared with the corresponding peptides containing Leu residue. The results show that the helix driving capacity of l and d forms of t-Leu is less than that of Leu residue. In poly t-Leu peptides, the population of collagen/inverse collagen-type structures or right/left handed-helical structures for l and d forms respectively is found to be chain length-dependent. The stability of the helical structures is increased by ~2 kcal per residue over the collagen-type structure in poly t-Leu peptides with chain length greater than five residues. Molecular view of peptides in collagen-type structure shows that the bulky side chains of t-Leu residues mask the NH moieties of the peptide bond, while the carbonyl groups lying along the helical groove are accessible to the small solvent molecules. Molecular model building suggests that one ethylene glycol molecule interacts by forming hydrogen bonds with carbonyl groups of two adjacent t-Leu residues. To the best of our knowledge, this is the first study of its own kind on the construction of a single-strand collagen/inverse collagen-type structure using unusual amino acid residues. Such synthetic collagen mimetic peptides shall exhibit specific affinity to natural collagen under controlled thermal conditions (heat or laser treatment) and hence can be explored as a new targeting method to attach therapeutic drugs to collagens in the living tissues and to biomaterials that incorporate natural collagens.

Keywords: l/d t-Leu helical tendency, Polytheonamide B, Peptide designing, Helix without hydrogen bonds, Single-strand collagen

*E-mail: fatehhar@pu.ac.in; fateh_nandel@yahoo.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, April 2007, pp. 114-121

 

 

Exploring QSAR of peripheral benzodiazepine receptor binding affinity of N,N-dialkyl-2-phenylindol-3-yl-glyoxylamides using physico-chemical descriptors

Kunal Roy* and Manoj Kumar Dalai

Drug Theoretics & Cheminformatics Lab, Division of Medicinal & Pharmaceutical Chemistry,
Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700 032, India

Received 19 April 2006; revised 28 November 2006

The present QSAR study has attempted to explore the structural and physicochemical requirements of ligands N,N-dialkyl-2-phenylindol-3-yl-glyoxylamides for binding with peripheral benzodiazepine receptor (PBR). The calcu­lated partition coefficient values show parabolic relations with the PBR binding affinity, suggesting that the binding affinity increases with increase in the partition coefficient of the compounds until it reaches the critical value after which the affinity decreases. The critical value of logP is within range of 6.052-6.410. Furthermore, positive Wang-Ford charge values of carbonyl oxygens of the glyoxamide moiety and negative Wang-Ford charge value of the glyoxamide nitrogen are conducive for the binding affinity. Again, the indole moiety should have favorable charge distribution. Higher values of the parameters dipole moment (Dipole) and moment of inertia (I_z) of the ligands are conducive for the binding affinity. The presence of hydrogen atom at R2 and cyclic moiety at R1 and R2 positions are detrimental to the binding affinity.

Keywords: QSAR, Peripheral benzodiazepine receptor, N,N-Dialkyl-2-phenylindol-3-yl-glyoxylamides, Physico-chemical descriptors, Binding affinity

*E-mail: kunalroy_in@yahoo.com,

  Note

 

 

Indian Journal of Biochemistry & Biophysics

Vol 44, April, 2007, pp. 122-125

 

 

Purification of protein from a crude mixture through SDS-PAGE transfer method

Dipankar Bhattacharyya, Arindam Basu and Parimal C Sen*

Department of Chemistry, Bose Institute, 93/1, A.P.C. Road, Kolkata 700 009, India

Received 06 July 2006; revised 27 February 2007

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) transfer method was used for purification and enrichment of the protein from crude sample. Coomassie blue/ZnSO4 stained protein band(s) containing intact polyacrylamide gel were loaded on to another polyacrylamide gel either alone or as pooled gel bands. Two/three bands were combined together and arranged tightly over one another, sealed with stacking gel and ran in another gel, which was quite useful for enrichment and purification of a particular protein from a complex mixture. Recovery of protein by gel transfer method was found to be 70% in case of ZnSO4 staining, whereas around 30% recovery was possible, following Coomassie blue staining. The method described here for purification of protein(s) from a complex mixture, following gel transfer procedure could be useful for further characterization of the desired protein.

Keywords: SDS-PAGE, Coomassie blue and ZnSO4 staining, Gel transfer, Protein purification

*Email: senpc03@yahoo.com; parimal@bosemain.boseinst.ac.in

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

AUTHOR INDEX

 

 


Akella S

82

 

 

Basu A

122

Bhattacharyya D

122

 

 

Chatterjee B P

94

Chatterjee U

94

Cheng X

101

 

 

Dalai M K

114

Divya Shree A N

71

 

 

Katyare S S

88

 

 

Mitra C K

82

Modi H R

88

Mondal G

94

 

 

Nandel F S

106

 

 

Patel M A

88

Patel S P

88

Poorani N

76

 

 

Ramalingam

76

Roy K

114

 

 

Sadasivam S

76

Saini A

106

Samanta S

94

Saraswathy N

76

Sen P C

122

Subha K

76

Subramanian S

71

 

 

Wang C

101

 

 

Xi Z

101

Xue Z

101

 

 

Yang L

101

 

 

Zhang X

101